RELATED APPLICATIONS
FIELD
[0002] The present invention, in some embodiments thereof, relates to methods and devices
for determining a disease state in a patient. In some embodiments, human serum albumin
may be analyzed for associated molecules, wherein the associated molecules are related
to a disease such as cancer.
BACKGROUND
[0003] There are two considerations generally relating to cancer: it is the number one killer
in the United States (when taken as an aggregate of the multiple forms of the disease)
and the earlier the detection the greater the likelihood for a positive outcome. Cancers
are often discovered only when they are large enough to be seen with an imaging device
or when they have spread so much that they have severely affected the health of the
ill patient.
[0004] Cancer diagnostics suffer from several major challenges. Some tests, notably PSA
(prostate-specific antigen) tests for prostate cancer, are often inaccurate harbingers
of the disease's presence. Additionally, some tests require unpleasant surgery or
the like to obtain biopsies. Other tests, notably the BRAC series of genetic tests
for breast cancer, are prohibitively expensive. An ideal cancer diagnostic would require
nothing more than a blood sample and would give highly accurate and reliable results,
even for "small" cancers that have not yet reached a size easily visible on X-ray,
CAT, and MRI machines.
[0005] The prior art generally describes methods for determining a disease state directly
from the presence of a predetermined biomolecule produced as a result of said disease
state. Improvements in determining disease states, including but not limited to cancers,
are thus needed.
SUMMARY
[0006] The present invention, in some embodiments thereof, relates to methods and devices
for determining a disease state in a patient. In some embodiments, human serum albumin
may be analyzed for associated molecules, wherein the associated molecules are related
to a disease such as cancer. The subject matter of the present invention involves,
in some cases, interrelated products, alternative solutions to a particular problem,
and/or a plurality of different uses of one or more systems and/or articles.
[0007] It is therefore a purpose of the present invention, in some embodiments, to describe
methods and devices for determining a disease state as a function of the interaction
behavior of a blood component, such as human serum albumin (HSA). In some embodiments
of the invention, the interaction of HSA with at least one biomolecule related to
a disease state may cause HSA to partition differently in a biphasic aqueous system,
as compared to HSA from blood of a healthy individual.
[0008] The invention includes, in certain embodiments, a method for diagnosing breast cancer,
other cancers, or other disease states. In one embodiment, the method comprises the
following: collecting blood from a patient; separating serum or plasma from the blood;
partitioning the serum or plasma in at least one aqueous two-phase partitioning system;
assaying aqueous phases of the at least one two phase partitioning system for human
serum albumin (HSA) using at least one assay specific for the albumin; calculating
or determining the partition coefficient K of albumin for each aqueous phase of the
two phase partitioning system; and, determining presence or risk level of breast cancer
or other cancer in the patient by comparing numerical values of calculated partition
coefficients with reference values previously determined for HSA in serum or plasma
taken from individuals with and without breast cancer or other cancers.
[0009] In one aspect of the method, the assaying is performed with an HSA-specific immuno-based
assay.
[0010] In another aspect of the method, the assaying is performed with an assay that is
specific for a biomolecule that is associated with HSA.
[0011] In another aspect of the method, the two-phase partitioning system is adapted to
differentially partition albumin when cancer is present or absent in the patient.
[0012] In another aspect of the method, the method is applied in conjunction with a mammogram,
genetic, or other cancer test.
[0013] In another aspect of the method, the method is applied as a part of a mathematical
or statistical algorithm, in conjunction with information obtained from a mammogram,
genetic, or other cancer test.
[0014] In another aspect of the method, the individuals without cancer include individuals
with benign tumors.
[0015] In another aspect of the method, the diagnosing is used to screen, diagnose, classify
according to phenotype/genotype, aid in therapeutic course of action, monitor progression,
or detect recurrence of cancer.
[0016] In another aspect of the method, the diagnosis is performed by comparing the value
of the partition coefficient, K, to its prior value or values at prior time or times
of the same individual.
[0017] In another aspect of the method, the numerical value of the partition coefficient
is used to select a therapeutic drug.
[0018] In another aspect of the method, the partitioning involves vortexing and centrifugation
of the two-phase partitioning system with human serum albumin present.
[0019] In another aspect of the method, there is an additional step of removing at least
one biomolecule from the human serum albumin.
[0020] In another aspect of the method, the at least one peptide is characterized for use
as a biomarker for cancer presence or risk.
[0021] In another aspect of the method, the reference values are determined from blood samples
taken from individuals with and free of cancer.
[0022] The invention also provides, in some embodiments, a device for the detection of breast
cancer or other cancers or disease states. In some cases, the device includes: a unit
for collecting blood from a patient and separating serum or plasma from the blood;
at least one aqueous two-phase partitioning system; a unit for partitioning a portion
of the serum or plasma in the two phase partitioning system; an assay for determining
the presence of human serum albumin in aqueous phases of the two phase partitioning
system; a computing element adapted to determine a coefficient K, wherein K represents
the distribution of human serum albumin in the aqueous portions of the two-phase partitioning
system; and, a determination element adapted to compare the coefficient K with known
values of K for blood samples of healthy individuals and individuals with breast cancer
or other cancers.
[0023] In one aspect of the device, the assay is realized as an HSA-specific immuno-based
assay.
[0024] In another aspect of the device, there is additionally a microfluidics device.
[0025] In another aspect of the device, the two-phase partitioning system is adapted to
differentially partition albumin when cancer is present or absent in the patient.
[0026] In another aspect of the device, the computing element and the determination element
are realized as a single element associated with a computing device.
[0027] In another aspect of the device, the computing device is realized as one of the following:
mainframe computer, laptop computer, tablet computer, mobile computing device, and
tabletop computer.
[0028] In another aspect of the device, the unit for partitioning includes a centrifuge
component.
[0029] In another aspect of the device, the microfluidics device does not include vortexing
or centrifugation in the unit for partitioning.
[0030] The invention additionally includes, in some embodiments, a method for diagnosing
a disease in a patient. The method, in some cases, comprises the following: collecting
blood from the patient; separating serum or plasma from the blood; partitioning the
serum or plasma in at least one aqueous two-phase partitioning system, wherein the
two-phase partitioning system is adapted to differentially partition albumin when
the disease is present or absent in the patient; assaying aqueous phases of the at
least one two phase partitioning system for human serum albumin (HSA) using specific
assay for the albumin; calculating or determining partition coefficient K of albumin
for each aqueous phases; and, determining presence of the disease in the patient by
comparing numerical values of calculated partition coefficients with reference values
previously determined for albumin in serum or plasma taken from individuals with and
without the disease.
[0031] In one aspect of the method, the disease is cancer.
[0032] In another aspect of the method, the cancer is selected from the following: throat
cancer, stomach cancer, pancreatic cancer, brain cancer, lung cancer, cervical cancer,
prostate cancer, breast cancer, testicular cancer, ovarian cancer, oral cancer, throat
cancer, esophagus cancer, and intestinal cancer and intestinal cancer.
[0033] In another aspect of the method, the disease is realized as a plurality of diseases.
[0034] In another aspect of the method, the disease is hereditary.
[0035] In another aspect of the method, the partition coefficients obtained from a plurality
of aqueous two-phase systems are combined using mathematical techniques into a numerical
signature.
[0036] In another aspect of the method, the numerical signature is compared with numerical
signatures obtained from reference values and is used for diagnosis.
[0037] Still another aspect of the present invention is generally directed to a method for
diagnosing breast cancer. In one set of embodiments, the method comprises acts of
collecting blood from a patient, separating serum or plasma from said blood, partitioning
said serum or plasma in at least one aqueous two-phase partitioning system, assaying
aqueous phases of said at least one two phase partitioning system for human serum
albumin (HSA) using at least one assay specific for said albumin, calculating partition
coefficient K of albumin for each aqueous phase of said two phase partitioning system,
and determining presence, lack of, or risk level of breast cancer in said patient
by comparing numerical values of calculated partition coefficients with reference
values previously determined for HSA in serum or plasma taken from individuals with
and without breast cancer.
[0038] In another set of embodiments, the method includes acts of collecting blood from
a patient, separating serum or plasma from said blood, partitioning said serum or
plasma in at least one aqueous two-phase partitioning system, assaying aqueous phases
of said at least one two phase partitioning system for human serum albumin (HSA) using
at least one assay specific for said albumin, calculating partition coefficient K
of albumin for each aqueous phase of said two phase partitioning system, and determining
presence, lack of, or risk level of cancer in said patient by comparing numerical
values of calculated partition coefficients with reference values previously determined
for HSA in serum or plasma taken from individuals with and without cancer.
[0039] The method, in another aspect, is generally directed to a method for diagnosing a
disease in a patient. In one set of embodiments, the method includes acts of collecting
blood from said patient, separating serum or plasma from said blood, partitioning
said serum or plasma in at least one aqueous two-phase partitioning system, wherein
said two-phase partitioning system is adapted to differentially partition albumin
when said disease is present or absent in said patient, assaying aqueous phases of
said at least one two phase partitioning system for human serum albumin (HSA) using
specific assay for said albumin, calculating partition coefficient K of albumin for
each aqueous phases, and determining presence of said disease in said patient by comparing
numerical values of calculated partition coefficients with reference values previously
determined for albumin in serum or plasma taken from individuals with and without
said disease.
[0040] In yet another aspect, the method is generally directed to acts of partitioning a
sample arising from a subject in an aqueous two-phase partitioning system, and determining
the distribution of human serum albumin within the phases of the two-phase partitioning
system.
[0041] Still another aspect of the present invention is generally directed to a device.
According to one set of embodiments, the device is a device for the detection of breast
cancer. In one set of embodiments, the device comprises a unit for collecting blood
from a patient and separating serum or plasma from said blood, at least one aqueous
two-phase partitioning system in fluid communication with the unit for collecting
blood, a unit for partitioning a portion of said serum or plasma in said two phase
partitioning system in fluid communication with the partitioning system, an assay
for determining the presence of human serum albumin in aqueous phases of said two
phase partitioning system, a computing element adapted to determine a coefficient
K, wherein K represents the distribution of human serum albumin in the aqueous portions
of said two-phase partitioning system, and a determination element adapted to compare
said coefficient K with known values of K for blood samples of healthy individuals
and individuals with breast cancer.
[0042] In another set of embodiments, the device is a device for the detection of cancer.
In certain embodiments, the device comprises a unit for collecting blood from a patient
and separating serum or plasma from said blood, at least one aqueous two-phase partitioning
system in fluid communication with the unit for collecting blood, a unit for partitioning
a portion of said serum or plasma in said two phase partitioning system in fluid communication
with the partitioning system, an assay for determining the presence of human serum
albumin in aqueous phases of said two phase partitioning system, a computing element
adapted to determine a coefficient K, wherein K represents the distribution of human
serum albumin in the aqueous portions of said two-phase partitioning system, and a
determination element adapted to compare said coefficient K with known values of K
for blood samples of healthy individuals and individuals with cancer.
[0043] Other advantages and novel features of the present invention will become apparent
from the following detailed description of various non-limiting embodiments of the
invention when considered in conjunction with the accompanying figures. In cases where
the present specification and a document incorporated by reference include conflicting
and/or inconsistent disclosure, the present specification shall control. If two or
more documents incorporated by reference include conflicting and/or inconsistent disclosure
with respect to each other, then the document having the later effective date shall
control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Non-limiting embodiments of the present invention will be described by way of example
with reference to the accompanying figures, which are schematic and are not intended
to be drawn to scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For purposes of clarity,
not every component is labeled in every figure, nor is every component of each embodiment
of the invention shown where illustration is not necessary to allow those of ordinary
skill in the art to understand the invention. In the figures:
FIG. 1 shows a flowchart related to a method of one embodiment of the present invention;
FIG. 2 shows a flowchart of method associated with another embodiment of the invention;
FIG. 3 shows a schematic view of yet another embodiment of the invention;
FIG. 4 shows a method associated with another embodiment of the invention; and,
FIG. 5 shows a schematic view of one embodiment of the invention.
FIG. 6 shows a dot histogram corresponding to the data in Table 2 for early stage
breast cancer and normal/benign groups, according to another embodiment of the invention;
FIG. 7 shows a Receiver-Operating Characteristic plot of the data in Table 2, in one
embodiment;
FIG. 8 shows the differences between the value of the partition coefficient of HSA
for breast cancer samples in Table 4, and non-cancerous samples, including benign
breast cancer and other cancers and benign conditions, in another embodiment; and
FIG. 9 shows human serum albumin.
DETAILED DESCRIPTION
[0045] The present invention, in some embodiments thereof, relates to methods and devices
for determining a disease state in a patient. In some embodiments, human serum albumin
may be analyzed for associated molecules, wherein the associated molecules are related
to a disease such as cancer. Certain aspects of the invention are generally directed
to devices and methods for determining a disease state as a function of the three-dimensional
structure of a blood protein, human serum albumin (HSA) or its complex with other
ligands, e.g., due to binding to disease-specific ligands. HSA interacts with biomolecules
associated with a disease presence, and liganded HSA may differentially partition
between aqueous phases of a predetermined partitioning system, wherein the partitioning
behavior differs between HSA from healthy individuals and HSA from people harboring
the predetermined disease, such as breast cancer.
[0046] For instance, in some embodiments, the present invention is generally related to
methods and devices for detecting the presence or risk of acquiring a disease state
as related to differential solubility behavior of human serum albumin (HSA) as it
is associated with ligands related to said disease state. Aqueous partitioning systems
are employed wherein the partitioning of liganded HSA is differentiated between healthy
and disease states.
[0047] HSA, being the most abundant blood protein and the major carrier of both small and
large compounds, is typically not considered as a useful source of information for
assessment of a physiological state or condition of a subject. Indeed, HSA is usually
first removed from serum or plasma before analysis, since the concentration of HSA
is much greater than signaling or other proteins of interest. In some cases, excess
HSA may be present in a blood sample, such as serum or plasma, by up to 11 orders
of magnitude over other proteins of interest, thus significantly potentially masking
other proteins during analysis. Accordingly, HSA is typically seen as undesirable
for biomarker discover or analysis.
[0048] Furthermore, as HSA is present in the blood in high concentrations compared to other
proteins, HSA often interacts with almost everything in the blood or the body, e.g.,
to a degree that significantly masks other proteins of interest. Accordingly, due
to its ubiquity and its ability to interact with other components in the blood, HSA
has not generally been considered to be useful in the analysis of any specific diseases
or conditions, such as cancer. Instead, as noted above, HSA is usually ignored or
filtered out of the blood. Accordingly, it is surprising that HSA would be of any
utility or interest in diagnosing specific medical diseases or conditions, such as
breast cancer or other cancers or diseases such as those discussed in greater detail
below.
[0049] For purposes of better understanding some embodiments of the present invention, as
illustrated in Figures 1-8 of the drawings, reference is first made to FIG. 1. FIG.
1 shows a flowchart of a method of one embodiment of the invention. The method steps
include, in this example, the following: collecting blood from a patient; separating
serum or plasma from the blood; optionally performing additional fractionation and/or
sample preparation steps to further separate certain species; partitioning the serum
or plasma in at least one aqueous two-phase partitioning system; assaying aqueous
phases of the at least one two phase partitioning system for human serum albumin (HSA)
using at least one assay specific for the albumin; calculating or determining partition
coefficient K of albumin for each aqueous phase of the two phase partitioning system;
and, determining presence or risk level of breast cancer in the patient by comparing
numerical values of calculated partition coefficients with reference values previously
determined for HSA in serum or plasma taken from individuals with and without breast
cancer. This allows for detection of or determination of risk for breast cancers,
or other cancers, via a traditional blood sample.
[0050] In some cases, blood is collected and the serum or plasma is isolated and analyzed.
Human serum albumin is the most abundant protein in the serum. HSA has a tendency
to interact with or bind proteins, peptides or other biological ligands, serving as
carrier protein for many small and large molecules. These biomolecules may come from
myriad cellular sources and their specific functions may be unknown. However, since
HSA half-life in circulation is over 20 days, its role as carrier protein may allow
circulating disease-associated peptides and other molecules to accumulate in time.
Thus, even small amounts of a disease-associated biomolecules, e.g., from a small
tumor, which continuously enter the circulation, could be amplified in time by binding
to HSA, thus avoiding clearance mechanisms. Accordingly, in some embodiments, a blood
sample (or other suitable fluid sample) containing HSA is collected from a patient.
Any suitable technique known to those of ordinary skill in the art may be used to
collect blood or other suitable fluids.
[0051] In some embodiments, HSA in the blood may be partitioned between aqueous phases in
a predetermined biphasic (or higher, i.e., multiphasic) liquid system. The HSA enters
one or more of the phases, but in general not at equal levels, e.g., due to differences
in the aqueous solvent characteristics in the two phases. Without wishing to be bound
by any theory, it is believed that the specific amount of HSA entering a phase may
be further related to associated ligands on the HSA. In some cases, the specific ligands
associated with the HSA may not even be known, identified, or understood; however,
differences in HSA can still be readily determined, e.g., causing different partitioning
between the aqueous phases. For instance, the HSA dissolved in each phase may vary
between healthy (including benign) patients and those with breast cancer. In addition,
it should be understood that although breast cancer is used here, this is by way of
example only, and other cancers may also be determined, not just breast cancer. The
subject may be male or female, as both sexes can be afflicted by a variety of cancers,
including but not limited to breast cancers. The subject is typically human, although
non-human animals may be studied in certain embodiments (e.g., using the appropriate
species instead of human serum albumin, e.g., bovine serum albumin in the case of
cow).
[0052] Thus, in some embodiments, the aqueous-based liquids used in the partitioning system
are pre-selected to provide this differentiation of HSA behavior as a function of
presence or absence of cancers (or the risk of cancers), such as breast cancers, in
a patient. In some cases, the specific form of cancer may not necessarily be readily
identified, e.g., a cancer may nevertheless cause changes in HAS partitioning, even
if the cancer itself is not identified.
[0053] Once the HSA has been partitioned, in steps that may involve, e.g., vortexting, mixing,
centrifugation, or other manipulations, the HSA in each phase may be assayed using
techniques known to those of ordinary skill in the art. Examples include, but are
not limited to, immuno-specific assays like ELISA. Such assays may be directed towards
a ligand or a plurality of ligands associated with the HSA, if it is known, although
this is not always required. With quantification of HSA or its associated ligands
in both phases after partitioning, one may determine a ratio of the amount of HSA
(or ligands) in each phase. This ratio, K, may vary significantly in some cases between
HSA taken from healthy patients and those with cancers or high risk for cancers, e.g.,
with pre-cancerous growths. For instance, patients with an active breast cancer or
those with a high risk for the disease may be determined in various embodiments. In
some cases, K may be compared to data previously collected for known healthy and known
ill patients, or compared with data collected earlier from the same patient presumably
in a healthy state. The K value may be determined as falling within values for healthy
patients or within values for those known to have breast or other cancers, or a high
risk factor for such cancers, etc. Since the individual disease biology may differ
for each individual, conventional statistical techniques known to those of ordinary
skill in the art used in the study of diagnostics, such as Receiver-Operating Characteristics,
may be used to define a cut-off point for K, depending on the desired levels of sensitivity
and specificity.
[0054] The ligands associated with HSA which allow for the differentiation may never be
known, in some cases. Moreover, since cancer is a heterogeneous disease on the molecular
level, such methods, which focuses on differential solubility of the HSA carrier protein
that potentially binds a myriad of disease-associated molecules, may be advantageous
in comparison with the conventional approach of isolating a single "silver bullet"
biomarker. For instance, the ligands binding to HSA may, in reality, be composed of
a family of different ligands or biomarkers, and their aggregate effect on HSA may
be determined, even if the effect of any single biomarker is not completely determined.
In some embodiments, one may isolate said ligands for the purpose of assigning them
as biomarkers for certain cancers, such as breast cancer. Methods for removing biomolecules
from HSA and analyzing said biomolecules are those traditionally used in protein chemistry
work, and include chromatography, mass spectrometry, etc. Since one does not have
to initially identify and define biomarkers in this embodiment, a great deal of effort
may be saved instead by analyzing changes in HSA partitioning behavior as a function
of the presence or absence of cancer, or the risk of cancer, according to certain
embodiment of the invention.
[0055] The approximate sequence of human serum albumin is shown in Fig. 9. The italicized
first 24 amino acids are signal and propeptide portions not observed in the transcribed,
translated, and transported protein but present in the gene. There are 609 amino acids
in this sequence with only 585 amino acids in the final product observed in the blood.
[0056] Attention is now turned to FIG. 2, which shows a flowchart for a method associated
with another embodiment. The method steps include the following: collecting blood
from a patient; separating serum or plasma from the blood; partitioning the serum
or plasma in at least one aqueous two-phase partitioning system, wherein the two-phase
partitioning system is adapted to differentially partition albumin when the disease
is present or absent in the patient; assaying aqueous phases of the at least one two
phase partitioning system for human serum albumin (HSA) using specific assay for the
albumin; calculating or determining partition coefficient K of albumin for each aqueous
phases; and, determining presence of the disease in the patient by comparing numerical
values of calculated partition coefficients with reference values previously determined
for albumin in serum or plasma taken from the same individuals or from individuals
with and without the disease. This allows for detection of or determination of a predetermined
disease state via a traditional blood sample. Blood is collected and the serum or
plasma is analyzed. In some cases, HSA is partitioned between aqueous phases in a
predetermined biphasic (or higher) liquid system. The HSA enters both phases, but
in general not at equal levels due to differences in the aqueous solvent characteristics
in the two phases, e.g., as discussed above. The specific amount entering a phase
may be further being related to HSA having at least one associated ligand, wherein
the ligand has some relationship to the presence of the disease state in the patient.
Additionally, the HSA dissolved in each phase may vary between healthy (defined as
those lacking the specific disease in question) patients and those with the predetermined
disease.
[0057] The liquids used in the partitioning system may be pre-selected in some cases to
provide this differentiation of HSA behavior as a function of presence or absences
of said disease in a patient, or to determining the risk of the disease in the patient.
Once the HSA has been partitioned, in steps that may involve vortexing, centrifugation,
microfluidic transfer or other manipulations, the HSA in each phase may be assayed
using techniques known to those of ordinary skill in the art. Examples include, but
are not limited to, immuno-specific assays like ELISA. With identification of HSA
and its quantification, one may determine a ratio of HSA in each phase. This ratio,
K, may in some cases vary significantly between HSA taken from healthy patients and
those with the predetermined disease. K is thus compared to data previously collected
for known healthy and known ill patients. The K value will either fall within values
for healthy patients or within values for those known to have the disease in question.
Diseases that can be determined include, but are not limited to cancers, hereditary
diseases, bacterial infections, viral infections, and sepsis. In some cases, any disease
which causes any alteration in ligands that associate with HAS in the blood can be
determined, in accordance with various embodiments of the invention.
[0058] Preparation of partitioning systems for various aspects of the invention, including
those described above, may, in some cases, involves large-scale robotic screening
of liquid samples to determine which liquids provide analytical differentiation of
HSA from healthy samples and those who have the predetermined disease associated with
the assay under development. Once a solvent system, comprising two, three, or more
immiscible liquid layers, is defined, HSA from people of unknown health conditions
(e.g., having a disease such as cancer, or other diseases described herein) may be
partitioned, assayed, and analyzed. K values determined may be compared to known K
values for healthy people and K values for those known to be ill or at risk for the
predetermined disease, or compared with K values of the same individual at earlier
time (presumably at a healthy state).
[0059] One aspect of the present invention is generally directed to HSA. HSA is an abundant
blood protein. Without wishing to be bound by any theory, determining HSA in the blood,
e.g., through partitioning as discussed herein, may be useful for determining disease
states in an individual, e.g., through comparison with other healthy and/or diseased
individuals. In and of itself, HSA does not give much information on disease states
in the blood or in other organs of the body. Yet, it is the nature of HSA to bind
peptides, proteins and other biomolecules. The ensuing result of the binding behavior
would appear to be different between healthy and ill individuals as a function of
potential ligands available for HSA interaction: HSA isolated from the blood of ill
people appears to have different ligands than does HSA isolated from healthy people.
One advantage of the instant invention is that one does not have to identify, isolate,
or otherwise define a biomarker for a given disease or illness condition. Indeed,
it may be exceedingly difficult or even impossible to define a disease condition based
on a unique biomarker for complex, heterogeneous diseases such as cancer. Rather,
one may use HSA's proclivity for binding proteins and the ability to differentially
dissolve HSA in a plurality of solvents, e.g., due to its modified three or higher
dimensional structure when bound up with various ligands. Dissolution of HSA may be
different not only between solvents, but also different as a function of HSA liganded
with proteins and the like from a healthy person and HSA liganded with proteins and
the like from a patient harboring disease. Yet another advantage of focusing on HSA
and its bound biomolecules is related to its long life in circulation-over 20 days-which
may in some cases serve to amplify the effect of even small amounts of disease-associated
biomolecules that continuously enter the circulation over time.
[0060] Attention is turned to FIG. 3 which is directed to an example of a device. The components
of a device associated with the cancer detection system 300 include: a unit for collecting
blood 310 from a patient and separating 315 serum or plasma from the blood; at least
one aqueous two-phase partitioning system 320; a unit for partitioning 330 a portion
of the serum or plasma in the two phase partitioning system; an assay 340 for determining
the presence of human serum albumin 335 in aqueous phases of the two phase partitioning
system; a computing element 350 adapted to determine a coefficient K, wherein K represents
the distribution of human serum albumin 335 in the aqueous portions of the two-phase
partitioning system; and, a determination element 360 adapted to compare the coefficient
K with known values of K for blood samples of healthy individuals and individuals
with cancer. The unit for collecting blood may, for example, include a needle or syringe.
In some cases, one or more of these units may be contained within a single device.
[0061] It should be understood that all of the elements described in this embodiment may
be included in a single unit or a small number of modular components; the elements
are shown individually so as to aid in the understanding of the present invention.
For example, computing element 350 and determination element 360 may generally be
associated with a computing device 370 that may including a controller element (not
shown) that directs various tasks from the receipt of blood to producing a final determination
of the presence or absence of cancer, or the risk of cancer in some cases. Computing
device 370 may be realized as any relevant device and includes but is not limited
to computers, hand-held computers, tablet computers, cellular phones, laptop computers,
and tabletop computers, or other computing devices known to those of ordinary skill
in the art.
[0062] Attention is turned to FIG. 4 which shows a flowchart in accordance with one embodiment
of the instant invention. The flowchart shown in FIG. 4 includes the following: collecting
blood from a patient; separating serum or plasma from the blood; partitioning the
serum or plasma in at least one aqueous two-phase partitioning system; assaying one
aqueous phase of the at least one two phase partitioning system for human serum albumin
(HSA) using at least one assay specific for the albumin; calculating partition coefficient
K of albumin for each aqueous phase of the two phase partitioning system based on
the measured amount of HSA in the one aqueous phase; and, determining presence or
risk level of breast cancer in the patient by comparing numerical value of calculated
partition coefficients with reference values previously determined for albumin in
serum or plasma taken from individuals with and without breast cancer, or from the
same patient at different time. As previously discussed, breast cancer is shown here
as a non-limiting example; in other embodiments, other cancers or disease states may
be determined.
[0063] In one embodiment, determination of HSA concentration may be performed in only one
phase of a two-phase aqueous system (e.g., only the upper phase or only the lower
phase). In some cases, for instance, the total HSA concentration may be determined
prior to partitioning; by knowing the amount present in one phase and the total amount
originally present, then one can determine the amount of HSA in the remaining phase.
Alternatively, one may omit measurement of HSA concentration in the sample, as HSA
concentrations are generally known and one may use appropriate literature values for
such determinations, or make reasonable approximations, e.g., based on the patient's
age, sex, blood pressure, height, and/or weight, etc. Thus, only one phase for HSA
concentration may be determined or measured. K may be calculated based on the one
measured HSA concentration value and the other calculated HSA concentration value;
the K value may be compared to known values for cancerous and non-cancerous states
(or other disease states such as those described herein). In some cases, a medical
determination may be performed based on the determined K value.
[0064] In some embodiments, HSA is partitioned between aqueous phases in a predetermined
biphasic (or higher) liquid system. HSA enters both phases, but in general not at
equal levels due to differences in the aqueous solvent characteristics in the two
phases, e.g., as previously discussed. The specific amount entering a phase may in
some cases be further related to HSA having at least one associated ligand. Additionally,
the HSA dissolved in each phase varies between healthy (including benign) patients
and those with diseases, e.g., cancers such as breast cancer. The aqueous-based liquids
used in the partitioning system may in some embodiments be pre-selected to provide
this differentiation of HSA behavior as a function of presence or absence of breast
cancer in a patient. Once the HSA has been partitioned, in steps that may involve,
for example, vortexting, mixing, centrifugation or other manipulations, the HSA in
one phase may be assayed using techniques known to those of ordinary skill in the
art, e.g., immuno-specific assays like ELISA. Such assays alternatively may be directed
towards a ligand or a plurality of ligands associated with the HSA. With quantification
of HSA or its associated ligands, one may determine a ratio of the amount of HSA (or
ligands) in each phase as described above. This ratio, K, may vary significantly between
HSA taken from healthy patients and those either with an active disease or those with
a high risk for the disease, e.g., of cancers such as breast cancers. K may be compared
to data previously collected for known healthy and known ill patients, or compared
with previously measured values for the same patient. The K value may be determined
as falling within values for healthy patients or within values for those known to
have breast or other cancers, or a high risk factor for such cancers, etc. Since the
individual disease biology may differ for each individual, conventional statistical
techniques known to those of ordinary skill in the art used in the study of diagnostics,
such as Receiver-Operating Characteristics, may be used to define a cut-off point
for K, depending on the desired levels of sensitivity and specificity.
[0065] The ligands associated with HSA which allow for the differentiation may never be
known, in some cases. Moreover, since cancer is a heterogeneous disease on the molecular
level, such methods, which focuses on differential solubility of the HSA carrier protein
that potentially binds a myriad of disease-associated molecules, may be advantageous
in comparison with the conventional approach of isolating a single "silver bullet"
biomarker. For instance, the ligands binding to HSA may, in reality, be composed of
a family of different ligands or biomarkers, and their aggregate effect on HSA may
be determined, even if the effect of any single biomarker is not completely determined.
In some embodiments, one may isolate said ligands for the purpose of assigning them
as biomarkers for certain cancers, such as breast cancer. Methods for removing biomolecules
from HSA and analyzing said biomolecules are those traditionally used in protein chemistry
work, and include chromatography, mass spectrometry, etc. Since one does not have
to initially identify and define biomarkers in this embodiment, a great deal of effort
may be saved instead by analyzing changes in HSA partitioning behavior as a function
of the presence or absence of cancer, or the risk of cancer, according to certain
embodiment of the invention.
[0066] Attention is turned to FIG. 5 which shows yet another embodiment. In the example
of FIG. 5, the components of a device associated with the breast cancer detection
system 800 include: a unit for collecting blood 810 from a patient and separating
815 serum or plasma from the blood; at least one aqueous two-phase partitioning system
820; a co-current or countercurrent chromatographic channel for partitioning 830 a
portion of the serum or plasma in the two phase partitioning system; an assay 840
for determining the presence of human serum albumin 835 in aqueous phases of the two
phase partitioning system; a computing element 850 adapted to determine a coefficient
K, wherein K represents the distribution of human serum albumin 835 in the aqueous
portions of the two-phase partitioning system; and, a determination element 860 adapted
to compare the coefficient K with known values of K for blood samples of healthy individuals
and individuals with cancers, such as breast cancer. It should be understood that
all of the elements described in this embodiment may be included in a single unit
or a small number of modular components; the elements are shown individually in this
figure so as to aid in the understanding of the present invention.
[0067] The aqueous partition system 820 may be added to the chromatographic unit either
before or with the serum sample including HSA. The serum sample may be introduced
into the channel independently or together with one of the flowing phases, and HSA
partitions between the phases as it travels downstream. When the equilibrium amounts
of HSA are established between the phases, samples from one or both of the two phases
are obtained, and the amounts of HSA in each phase are assayed using conventional
techniques, and are used to calculate the partition coefficient, K, as described before.
Such devices may employ microfluidics elements, and could one way to provide point-of-care
capabilities for the present invention.
[0068] Computing element 850 and determination element 860 may generally be associated with
a computing device 870 that may including a controller element (not shown) that directs
all necessary tasks from the receipt of blood to producing a final determination of
breast cancer presence. Computing device 370 may be realized as any relevant device
and includes but is not limited to computers, hand-held computers, tablet computers,
cellular phones, laptop computers, and tabletop computers, or other computing devices
known to those of ordinary skill in the art.
[0069] As mentioned, a variety of cancers may be determined in a blood sample (or other
suitable fluid sample), e.g., by determining partitioning of HSA in an aqueous two-phase
partitioning system. Examples of cancers include, but are not limited to: breast,
prostate, lung, ovarian, colorectal, and brain cancer. Other non-limiting examples
of cancers include biliary tract cancer; bladder cancer; brain cancer including glioblastomas
and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer;
endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including
acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS-associated leukemias
and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen's disease
and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease
and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma;
ovarian cancer including those arising from epithelial cells, stromal cells, germ
cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas
including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma;
skin cancer including melanoma, Kaposi's sarcoma, basocellular cancer, and squamous
cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma,
teratomas, choriocarcinomas; stromal tumors and germ cell tumors; thyroid cancer including
thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma
and Wilms' tumor. Still other examples of cancers include lymphomas, sarcomas and
carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synovioma,
mesothelioma, lymphangioendotheliosarcoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile
duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical
cancer, testicular tumor, lung carcinoma, non-small cell lung carcinoma, small cell
lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic
leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,
monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic)
leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's
disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia
and heavy chain.
[0070] As discussed, HSA may be partitioned using an aqueous two-phase partitioning system
or an aqueous multi-phase partitioning system, in certain embodiments. Aqueous two-phase
systems may arise in aqueous mixtures of different water-soluble polymers or a single
polymer and a specific salt. When two certain polymers, e.g., dextran (Dex) and polyethylene
glycol (PEG), or a single certain polymer and a certain inorganic salt, e.g. polyvinylpyrrolidone
(PVP) and sodium sulfate, are mixed in water above certain concentrations, the mixture
separates into two immiscible aqueous phases. There is a discrete interfacial boundary
separating two phases, one rich in one polymer and the other rich in the other polymer
or inorganic salt. The aqueous solvent in both phases provides media suitable for
biological products. Two-phase systems can be generalized to multiple phase system
by using different chemical components, and aqueous systems with a dozen or more phases
have been mentioned in the literature.
[0071] When a solute is introduced into such a two-phase system, it distributes between
the two phases. Partitioning of a solute is characterized by the partition coefficient
K defined as the ratio between the concentrations of the solute in the two immiscible
phases at equilibrium. It was previously shown that phase separation in aqueous polymer
systems results from different effects of two polymers (or a single polymer and a
salt) on the water structure. As the result of the different effects on water structure,
the solvent features of aqueous media in the coexisting phases differ from one another.
The difference between phases can be demonstrated by dielectric, solvatochromic, potentiometric,
and partition measurements.
[0072] The basic rules of solute partitioning in aqueous two-phase systems were shown to
be similar to those in water-organic solvent systems. However, what differences do
exist in the properties of the two phases in aqueous polymer systems are very small
relative to those observed in water-organic solvent systems, as should be expected
for a pair of solvents of the same (aqueous) nature. Importantly, the small differences
between the solvent features of the phases in aqueous two-phase or multi-phase systems
can be modified so as to amplify the observed partitioning that results when certain
structural features are present, e.g., such as ligands on HSA.
[0073] It is known that the polymer and salt compositions of each of the phases depend upon
the total polymer and salt composition of an aqueous two-phase system. The polymer
and salt composition of a given phase, in turn, governs the solvent features of an
aqueous media in this phase. These features include, but are not limited to, dielectric
properties, solvent polarity, ability of the solvent to participate in hydrophobic
hydration interactions with a solute, ability of the solvent to participate in electrostatic
interactions with a solute, and hydrogen bond acidity and basicity of the solvent.
All these and other solvent features of aqueous media in the coexisting phases may
be manipulated by selection of polymer and salt composition of an aqueous two-phase
system. These solvent features of the media govern the sensitivity of a given aqueous
two-phase system toward a particular type of solvent accessible chemical groups in
the receptor. This sensitivity, type, and topography of the solvent accessible groups
in two different proteins, for example, determine the possibility of separating or
partitioning proteins such as HSA in a given aqueous two-phase system. One example
of aqueous two-phase partitioning is described in
U.S. Patent No. 6,136,960, hereby incorporated in its entirety.
[0074] Without wishing to be bound by any theory, it is believed that partitioning of a
biopolymer in aqueous two-phase systems depends on its three-dimensional structure
and type and topography of chemical groups exposed to the solvent. Changes in the
3-D structure of a receptor induced by some effect, e.g., by binding of a ligand binding
or by structural degradation, also change the topography of solvent accessible chemical
groups in the biomolecule or both the topography and the type of the groups accessible
to solvent. One result of these changes is an alteration in the partition behavior
of the biomolecule or the ligand-bound-receptor. As a result, by monitoring the partition
coefficient of an analyte, it is possible to detect a change in the state of a structure
for which a partition coefficient is already known.
[0075] Similarly, such changes may be detected using other methods which have an underlying
dependence upon the topography and/or the types of solvent accessible groups. Examples
of such other methods include, but are not limited to, column liquid-liquid partition
chromatography (LLCP), heterogeneous two-phase systems or a multiphase heterogeneous
system.
[0076] In cases where a method as in determining a coefficient which reflects a relative
partitioning, e.g., as in a partition coefficient, a single descriptor is obtained.
While the many different aqueous two-phase systems all differ in their sensitivity
toward various chemical groups, e.g., charged and non-polar groups, the presence of
a detectable difference between two conformational states, in the form of a change
in a partition coefficient, may result from a great many different mechanisms. As
such, a similar change in a single partition coefficient may reflect very dissimilar
conformational changes. In addition, in some cases, more than one partitioning coefficient
may be obtained, e.g., as in a signature. See, e.g.,
U.S. Pat. No. 7,968,350, incorporated by reference in its entirety.
[0077] As used herein, "signature" refers to a particular representation of desired information,
which can be defined as a set of relative measures of interaction described above
obtained from experiments with different interacting components. Typically, a signature
is used in place of more detailed information when the latter is difficult to obtain,
or when it is not necessary to completely describe such information in order to make
use of it. For example, fingerprinting individual people is a well-recognized technique
to uniquely identify an individual (to a reasonable certainty), providing a conveniently
obtained and conveniently dense information set instead of describing the individual
using other representations, e.g., genetic makeup, or by using exhaustively physical
description and other information.
[0078] An "interaction signature," a used herein, means a signature characteristic of interaction
of a species with at least one other species, optionally also characteristic of interaction
of either or both species with another species or an environment (medium) in which
the species exist and/or with which the species interact.. For example, an interaction
signature may characterize interaction of a species with one phase of a multi-phase
system, and with another phase of the multi-phase system, with an overall interaction
signature characteristic of the relative interaction of the species with the two (or
more) phases. As further examples, an interaction signature can be characteristic
of interaction of a species with any number of phases of a multi-phase system, and
interaction signatures can exist for interaction between and among a variety of species
and a variety of phases of a multi-phase system.
[0079] In accordance with the desired attributes of a signature, an information set could
be described by numbers, mathematical expressions, by visual representations or by
other means that are known to those of skill in the art. The particular choice of
how a signature is represented will primarily dependent upon the specific technique
that is used to obtain the surrogate information that is used to construct the signature
and on the manner in which the signature is ultimately going to be used. As will be
recognized by those of skill in the art, many techniques have been developed to condense
and convey information in a manner that would be suitable for use in establishing
a signature or set of signatures.
[0080] Mathematical techniques suitable for obtaining a useful signature are numerous. They
include, but are not limited to, linear or nonlinear mapping (e.g., artificial neural
networks and partial least squares regression), matrix rotation and projection (e.g.,
principal component analysis and singular value decomposition), direct modeling using
differential equations that reflect the underlying physical process, if the underlying
physical process is known. Sometimes visual representations form superior signatures,
especially if they readily convey the desired information, e.g., differences amongst
individual sets, using shapes and colors which are easily conveyed to the observer.
[0081] Information sets to be used in generating signatures can comprise data from two or
more trial or experimental conditions, e.g., two or more partition coefficients. In
preferred embodiments, the method will use greater than 2, 3, 4, 5, 6, 8, 10 or 20
different sets of conditions. As will be recognized, the actual measurement or technique
used to obtain a measurement is not limited to a specific technique. As described
herein by way of example, the data can include partition coefficients. Other types
of data and the means for obtaining the data therefor will also be recognized by those
of skill in the art.
[0082] As mentioned, aqueous two-phase partitioning can be used to gather information for
generating a signature. Aqueous two-phase partitioning described in
U.S. Patent No. 6,136,960, hereby incorporated in its entirety, is one method by which information can be obtained
for generating a signature. Partitioning of a biopolymer in aqueous two-phase systems
depends on its three-dimensional structure and type and topography of chemical groups
exposed to the solvent. Changes in the 3-D structure of a receptor induced by some
effect, e.g., by binding of a ligand binding or by structural degradation, also change
the topography of solvent accessible chemical groups in the biomolecule or both the
topography and the type of the groups accessible to solvent. One result of these changes
is an alteration in the partition behavior of the biomolecule or the ligand-bound-receptor.
As a result, by monitoring the partition coefficient of an analyte such as HSA, it
is possible to detect a change in the state of a structure for which a partition coefficient
is already known.
[0083] "Aqueous," as used herein, refers to the characteristic properties of a solvent/solute
system wherein the solvating substance has a predominantly hydrophilic character.
Examples of aqueous solvent/solute systems include those where water, or compositions
containing water, is the predominant solvent.
[0084] "Aqueous multi-phase system," as used herein, refers to an aqueous system which consists
of greater than one aqueous phase in which an analyte species can reside, and which
can be used to characterize the structural state of the analyte species according
to the methods described herein. For example, this includes aqueous system which can
separate at equilibrium into two, three, or more immiscible phases. Aqueous multi-phase
systems are known in the art and this phrase, as used herein, is not meant to be inconsistent
with accepted meaning in the art.
[0085] An "interacting component" means a component, such as a phase of an aqueous multi-phase
system, that can interact with a species and provide information about that species
(for example, an affinity for the species). Multiple interacting components, exposed
to a species, can define a system that can provide a "relative measure of interaction"
between each component and the species. An interacting component can be aqueous or
non-aqueous, can be polymeric, organic (e.g. a protein, small molecule, etc.), inorganic
(e.g. a salt), or the like, or any combination. A set of interacting components can
form a system useful in and in part defining any experimental method which is used
to characterize the structural state of a species such as an analyte species according
to the methods described herein. Typically, a system of interacting components can
measure the relative interaction between the species and at least two interacting
components. An aqueous multi-phase system is a species of a system of interacting
components, and it is to be understood that where "Aqueous system" or "Aqueous multi-phase
system" is used herein, this is by way of example only, and any suitable system of
interacting components can be used.
[0086] Both aqueous two-phase and aqueous multi-phase systems, as used herein, also refer
to systems analogous to those comprising only aqueous solutions or suspensions. For
example, an aqueous two-phase system can include non-aqueous components in one or
more phases that are not liquid in character. In this aspect, aqueous phase systems
also refers to related techniques that rely on differential affinity of the biomolecule
to one media versus another, wherein the transport of the biomolecule between one
medium and, optionally, another medium occurs in an aqueous environment. Examples
of such "heterogeneous phase systems" include, but are not limited to, HPLC columns
or systems for liquid-liquid partition chromatography as are known to those of skill
in the art.
[0087] "Partition coefficient," as used herein, refers to the coefficient which is defined
by the ratio between the concentrations of the solute in the two immiscible phases
at equilibrium. For example, the partition coefficient (K) of an analyte in a two-phase
system is defined as the ratio of the concentration of analyte in the first phase
to that in the second phase. For multi-phase systems, there are multiple partition
coefficients wherein each partition coefficient defines the ratio of analyte in first
selected phase and a second selected phase. It will be recognized that the total number
of partition coefficients in any multi-phase system will be equal to the total number
of phases minus one.
[0088] "Bind," as used herein, means the well understood receptor/ligand binding as well
as other nonrandom association between an a biomolecule and its binding partner. "Specifically
bind," as used herein describes a binding partner or other ligand that does not cross
react substantially with any biomolecule other than the biomolecule or biomolecules
specified.
[0089] Generally, molecules which preferentially bind to each other are referred to as a
"specific binding pair." Such pairs include, but are not limited to, an antibody and
its antigen, a lectin and a carbohydrate which it binds, an enzyme and its substrate,
and a hormone and its cellular receptor. As generally used, the terms "receptor" and
"ligand" are used to identify a pair of binding molecules. Usually, the term "receptor"
is assigned to a member of a specific binding pair, which is of a class of molecules
known for its binding activity, e.g., antibodies. The term "receptor" is also preferentially
conferred on the member of a pair that is larger in size, e.g., on lectin in the case
of the lectin-carbohydrate pair. However, it will be recognized by those of skill
in the art that the identification of receptor and ligand is somewhat arbitrary, and
the term "ligand" may be used to refer to a molecule which others would call a "receptor."
The term "anti-ligand" is sometimes used in place of "receptor."
[0090] "Biomolecule," as used herein, means; peptides, polypeptides, proteins, protein complexes,
nucleotides, oligonucleotides, polynucleotides, nucleic acid complexes, saccharides,
oligosaccharides, carbohydrates, lipids and combinations, derivatives and mimetics
thereof.
[0091] "Detectable," as used herein, refers the ability of a species and/or a property of
the species to be discerned. One method of rendering a species detectable is to provide
further species, that bind or interact with the first species, that comprise a detectable
label. Examples of detectable labels include, but are not limited to, nucleic acid
labels, chemically reactive labels, fluorescence labels, enzymic labels and radioactive
labels.
[0092] Aqueous two-phase systems arise in aqueous mixtures of different water-soluble polymers
or a single polymer and specific salts. For example, dextran and polyethylene glycol
("PEG") are mixed in water above certain concentrations, the mixture separates into
two immiscible aqueous phases separated by a clear interfacial boundary. These two
separated phases are said to have resolved. In one phase, the solution is rich in
one polymer and, on the other side of this boundary in a second phase, the solution
is rich in the other polymer. The aqueous solvent in both phases provides media suitable
for biological products such as proteins or for other biomolecules.
[0093] Selection and modification of the types, as reflected in, for example, the chemical
nature, structure, and molecular weight, of the phase-forming polymers and the concentration
of the polymers can be used to vary the properties of the phases. In addition, the
composition of the phases can also be changed by the addition of inorganic salts and/or
organic additives. Changes to the composition of the phases can alter the properties
of the phases. Examples of types of aqueous two-phase systems that are useful for
detecting and/or characterizing the binding of a binding partner to a receptor include,
but are not limited to, dextran/PEG, dextran/polyvinylpyrrolidone, PEG/salt, and polyvinylpyrrolidone/salt.
[0094] Biomolecules such as proteins, nucleic acids or other also distribute between the
two phases when placed into such a system. This partitioning of a biomolecule between
the two phases is fairly simple. In some respects, it is similar to extraction as
is normally in the chemical arts. For example, in the case where phase-forming polymers
are used, solutions comprising one or more of the two polymers and the biomolecule
are mixed together such that both phase-forming polymers and the biomolecule are mixed.
The resulting solution is resolved and the two-phase system is formed. Optionally,
centrifugation can be used to enhance separation of the phases. It will be recognized
by those of skill in the art that partitioning behavior of a biomolecule may be influenced
by many variables, such as the pH, the polymers used, the salts used, other factors
relating to the composition of the system, as well as other factors such as temperature,
volume, etc. Optimization of these factors for desired effects can be accomplished
by routine practice by those of skill in the relevant arts in combination with the
current disclosure.
[0095] Evaluation of data from partitioning of a biomolecule can involve use of the partition
coefficient ("K"), which is defined as the ratio between the concentrations of the
biomolecule in the two immiscible phases at equilibrium. For example, the partition
coefficient, K, of a protein is defined as the ratio of the protein in first phase
to that in the second phase in a biphasic system. When multiple phase systems are
formed, there can be multiple independent partition coefficients that could be defined
between any two phases. From mass balance considerations, the number of independent
partition coefficients will be one less than the number of phases in the system.
[0096] It will be recognized that the partition coefficient K for a given biomolecule of
a given conformation will be a constant if the conditions and the composition of the
two-phase system to which it is subjected remain constant. Thus, if there are changes
in the observed partition coefficient K for the protein upon addition of a potential
binding partner, these changes can be presumed to result from changes in the protein
structure caused by formation of a protein-binding partner complex. "K", as used herein,
is used as specifically mathematically defined herein, and in all instances also includes,
by definition, any coefficient representing the relative measure of interaction between
a species and at least two interacting components.
[0097] In order to determine the partition coefficient K of a protein or a mixture of a
protein with another compound to be analyzed, concentrated stock solutions of all
the components (polymer 1, e.g., dextran; polymer 2, e.g., PEG, polyvinylpyrrolidone,
salts, etc.) in water can be prepared separately. The stock solutions of phase polymers,
salts, and the protein mixture can be mixed in the amounts and conditions (e.g., pH
from about 3.0 to about 9.0, temperature from about 4°C to 60 °C, salt concentration
from 0.001 to 5 mole/kg) appropriate to bring the system to the desired composition
and vigorously shaken. The system can then be allowed to equilibrate (resolve the
phases). Equilibration can be accomplished by allowing the solution to remain undisturbed,
or it can be accelerated by centrifugation, e.g., for 2-30 minutes at about 1000 to
4000 g or higher.
Aliquots of each settled (resolved) phase can be withdrawn from both the upper and
lower phases. The concentration of biomolecule can be determined for both the upper
and lower phases.
[0098] Different assay methods may be used to determine the concentration of the biomolecules
in each phase. The assays will depend upon the identity and type of biomolecule present.
Examples of suitable assay techniques include, but are not limited to, spectroscopic,
immunochemical, chemical, fluorescent, radiological and enzymatic assays. When the
biomolecule is a peptide or protein, e.g., HSA, the common peptide or protein detection
techniques can be used. These include direct spectrophotometry (monitoring the absorbance
at 280 nanometers) and dye binding reactions with Coomassie Blue G-250 or fluorescamine,
o-phthaldialdehyde, or other dyes and/or reagents. Alternatively, if the protein is
either an antibody or an antigen, immunochemical assays can also be used.
[0099] The concentration of the biomolecule(s) in each phase can then be used to determine
the partition coefficient, K, of the sample under the particular system conditions.
Since K reflects only the ratio of the two concentrations, the absolute values are
not typically required. It will be recognized that this can allow certain analytical
procedures to be simplified, e.g., calibration can be eliminated in some instances.
[0100] The partition coefficient can then be compared with other K values. For example,
a K value for a species can be compared to the K values for the species under different
conditions, a K value for a species can be compared to the K values for the species
when combined with other species, or a set of K values for a species can be compared
to other sets of K values.
[0101] Additionally, if the biomolecule concentration in the two-phase system of a fixed
composition is kept constant, the changes in the partition coefficient can be measured
not as changes in the partition coefficient value, but as those in the biomolecule
concentration in a given phase, for example, top phase of the system. This may be
more efficient than the determination of the partition coefficient value from measurements
of the biomolecule concentrations in each phase of a system.
[0102] Although the above has been described in conjunction with specific embodiments thereof,
it is evident that many alternatives, modifications and variations will be apparent
to those skilled in the art. Accordingly, it is intended to embrace all such alternatives,
modifications and variations that fall within the spirit and broad scope of the appended
claims. For example, certain embodiments of the present invention could be employed
for general population screening and one could use HSA as a general carrier unit for
detection of biomolecules not necessarily present in the blood: one could add exogenous
samples to HSA solutions and then partition the HSA as described above, with the goal
to detect a predetermined biomolecule.
[0103] The following documents are incorporated herein by reference in their entireties:
U.S. Pat. Nos. 8,099,242;
6,136,960; and
8,041,513. Also incorporated herein by reference in its entirety is
U.S. Patent Application Serial No. 14/312,907, filed June 24, 2014, entitled "Methods and Devices for Determining a Disease State."
[0104] The following examples are intended to illustrate certain embodiments of the present
invention, but do not exemplify the full scope of the invention.
EXAMPLE 1
[0105] In this example it is shown that albumin in human serum displays different partitioning
behavior when present in blood serum from a group of patients with malignant breast
tumor and a group of patients comprised of benign breast tumor and female healthy
donors. This example further demonstrates that different partitioning systems may
exhibit different levels of clinical specificity and degree of clinical differentiation
power.
[0106] Human serum samples corresponding to malignant and benign or healthy clinical phenotypes
were purchased from SeraCare Life Sciences. The diagnostic status of each sample was
additionally provided by SeraCare Life Sciences. Sample aliquots were obtained frozen
and stored at -80° C, and thawed and diluted 3-fold with water before introducing
to the aqueous two phase systems.
[0107] Aqueous two-phase system was prepared with Ficoll-70 (molecular weight of about 70,000),
Dex-70 (molecular weight of about 70,000), and 0.10 M sodium phosphate buffer, pH
7.4. The system in each tube was prepared by mixing the appropriate amounts of stock
polymer and buffer solutions dispensed by a liquid handling workstation Hamilton ML-4000
into a microtube of a total volume of 1.2 mL up to a total volume of a mixture of
393 microliters. A varied amount (15, 30, 45, 60, and 75 microliters) of each serum
sample and the corresponding amount (92, 77, 62, 47, and 32 microliters) of water
were added to a system. The ratio between the volumes of the two phases of each system
of a final volume of 500 microliters was as 1:1. The system was vigorously shaken
and centrifuged for 60 min at 3500 g in a refrigerated centrifuge with a microplate
rotor with the temperature maintained at 23 °C to speed the phase settling. Microtubes
were taken out of the centrifuge, and aliquots of 50 microliters from the top and
the bottom phases were withdrawn in duplicates and each diluted and mixed with appropriate
reagents as indicated below, and used for further analysis.
[0108] A second aqueous two-phase system was prepared with Ficoll-70 (molecular weight of
about 70,000), Dex-70 (molecular weight of about 70,000), and 0.15 M NaSCN, 0.15 M
Na
2SO
4, and 0.01 M sodium phosphate buffer, pH 7.4. The system in each tube was prepared
by mixing the appropriate amounts of stock polymer, salt, and buffer solutions dispensed
by liquid handling workstation Hamilton ML-4000 into a microtube of a total volume
of 1.2 mL up to a total volume of a mixture of 419 microliters. A varied amount (15,
30, 45, 60, and 75 microliters) of each serum sample and the corresponding amount
(70, 55, 40, 25, and 10 microliters) of water were added to a system. The ratio between
the volumes of the two phases of each system of a final volume of 504 microliters
was as 1:1. The system was vigorously shaken and centrifuged for 60 min at 3500 g
in a refrigerated centrifuge with a microplate rotor with the temperature maintained
at 23 °C to speed the phase settling. Microtubes were taken out of the centrifuge,
and aliquots of 40 microliters from the top and the bottom phases were withdrawn in
duplicates and each diluted and mixed with appropriate reagents as indicated below
and used for further analysis.
[0109] A third aqueous two-phase system was prepared with PEG-600 (polyethylene glycol with
molecular weight of about 600), Na
2SO
4, and sodium/potassium phosphate buffer, pH 7.4. The system in each tube was prepared
by mixing the appropriate amounts of stock polymer, salt, and buffer solutions dispensed
by liquid handling workstation Hamilton ML-4000 into a microtube of a total volume
of 1.2 mL up to a total volume of a mixture of 336 microliters. A varied amount (15,
30, 45, 60, and 75 microliters) of each serum sample and the corresponding amount
(149, 134, 119, 104, and 89 microliters) of water were added to a system. The ratio
between the volumes of the two phases of each system of a final volume of 500 microliters
was as 1:1. The system was vigorously shaken and centrifuged for 30 min at 3500 g
in a refrigerated centrifuge with a microplate rotor with the temperature maintained
at 23 °C to speed the phase settling. Microtubes were taken out of the centrifuge,
and aliquots of 40 microliters from the top and the bottom phases were withdrawn in
duplicates and each diluted and mixed with appropriate reagents as indicated below
and used for further analysis.
[0110] A fourth aqueous two-phase system was prepared with PEG-600 (polyethylene glycol
with molecular weight of about 600), Na
2SO
4, 0.15M NaCl, and sodium/potassium phosphate buffer, pH 7.4. The system in each tube
was prepared by mixing the appropriate amounts of stock polymer, salt, and buffer
solutions dispensed by liquid handling workstation Hamilton ML-4000 into a microtube
of a total volume of 1.2 mL up to a total volume of a mixture of 375 microliters.
A varied amount (15, 30, 45, 60, and 75 microliters) of each serum sample and the
corresponding amount (110, 95, 80, 65, and 50 microliters) of water were added to
a system. The ratio between the volumes of the two phases of each system of a final
volume of 500 microliters was as 1:1. The system was vigorously shaken and centrifuged
for 30 min at 3500 g in a refrigerated centrifuge with a microplate rotor with the
temperature maintained at 23 °C to speed the phase settling. Microtubes were taken
out of the centrifuge, and aliquots of 40 microliters from the top and the bottom
phases were withdrawn in duplicates and each diluted and mixed with appropriate reagents
as indicated below and used for further analysis.
[0111] Immunoassay analysis was conducted using aliquots (each of 40 microliters volume)
from the top and the bottom phases, diluted 10-fold with 0.9 wt. % NaCl solution in
water. The diluted aliquots were mixed by shaking and 200 microliters from each diluted
aliquot was transferred to sample cups of a clinical chemistry analyzer (Roche-Hitachi,
model 902). A turbidity-based microalbumin immunoassay was performed according to
the manufacturer's (Roche) protocol, and the albumin concentration in each aliquot
was recorded. The measured albumin concentration values in diluted aliquots from the
top phases were plotted as a function of the albumin concentration values of the similarly
diluted aliquots from the bottom phases. The partition coefficient for albumin was
determined as a slope of the plotted linear curve. While the partition coefficient
is defined as the ratio of the top to bottom phase concentrations, performing the
series dilution experiments as described herein and using the slope of the linear
regression line defines the partition coefficient value K to a greater accuracy, although
the two definitions are equivalent.
[0112] The partition coefficients for albumin are presented in Table 1 (below). The data
presented in Table 1 demonstrate that HSA partitioned in aqueous partitioning systems
could be used to classify clinical samples according to their phenotype source as
cancer or other (benign or normal). It was also demonstrated that aqueous partitioning
systems of different chemical compositions could be devised, and would provide different
degrees of capability for said classification ability. The final selection of an optimal
system could be done using a sufficiently statistically powered group of samples of
cancer and benign/normal origins, as defined using acceptable gold-standard diagnostics
techniques.
Table 1. Partition coefficients for albumin in serum samples from patients with diagnostic
status as indicated in different aqueous two-phase systems.
Partitioning System |
Partition coefficient K for albumin in serum from patients with |
|
Benign breast tumor or no tumor |
Malignant breast tumor |
Dextran-Ficoll- NaPB |
2.18 |
0.03 |
2.15 |
0.02 |
2.19 |
0.03 |
|
|
Dex-Ficoll-NaSCN-Na2SO4-NaPB |
0.330 |
0.009 |
0.29 |
0.011 |
0.342 |
0.005 |
0.29 |
0.010 |
PEG-600-Na2SO4-KNaPB |
2.13 |
0.02 |
2.39 |
0.036 |
2.19 |
0.02 |
2.25 |
0.041 |
PEG-600- Na2SO4-NaCl-KnaPB |
1.26 |
0.01 |
1.116 |
0.003 |
1.29 |
0.02 |
1.02 |
0.098 |
EXAMPLE 2
[0113] In this example it was demonstrated that the partition coefficients of albumin in
human serum from a group of patients with early (stage I) breast cancer are significantly
different from the partition coefficients of albumin in human serum from a second
group of patients with benign breast tumors.
[0114] Human serum samples corresponding to malignant and benign or healthy clinical phenotypes
were purchased from Proteogenix, Inc. The diagnostic status of each sample was also
provided by Proteogenix, Inc. Samples aliquots were obtained frozen and stored at
-80° C, and thawed and diluted 3-fold with water before introducing to the aqueous
two phase systems.
[0115] Aqueous two-phase system was prepared with PEG-1000 (polyethylene glycol with molecular
weight of about 1,000), Ficoll-70 (molecular weight of about 70,000), NaCl, and potassium
citrate buffer, pH 6.8. The system in each tube was prepared by mixing the appropriate
amounts of stock polymer and buffer solutions dispensed by a liquid handling workstation
Hamilton ML-4000 into a microtube of a total volume of 1.2 mL up to a total volume
of a mixture of 420 microliters. A varied amount (20, 30, 40, 50, 60, and 70 microliters)
of each serum sample and the corresponding amount (60, 50, 40, 30, 20, and 10 microliters)
of water were added to a system. The ratio between the volumes of the two phases of
each system of a final volume of 500 microliters was as 1:1. The system was vigorously
shaken and centrifuged for 60 min at 3500 g in a refrigerated centrifuge with a microplate
rotor with the temperature maintained at 23 °C to speed the phase settling. Microtubes
were taken out of the centrifuge, and aliquots of 40 microliters from the top and
the bottom phases were withdrawn in duplicates and each diluted and mixed with appropriate
reagents as indicated below and used for further analysis.
[0116] Immunoassay analysis was conducted with aliquots (each of 40 microliters volume)
from the top and the bottom phases, diluted 10-fold with 0.9 wt. % NaCl solution in
water. The diluted aliquots were mixed by shaking and 200 microliters from each diluted
aliquot was transferred to the sample cups of clinical chemistry analyzer (Roche-Hitachi,
model 902). The turbidity-based microalbumin immunoassay was performed according to
the manufacturer's (Roche) protocol, and the albumin concentration in each aliquot
was recorded. The measured albumin concentration values in diluted aliquots from the
top phases were plotted as a function of the albumin concentration values of the similarly
diluted aliquots from the bottom phases. The partition coefficient for albumin was
determined as a slope of the linear curve.
[0117] The partition coefficients for albumin in examined serum samples from patients with
diagnostic status indicated are presented in Table 2 (below) and illustrated in the
dot histogram of FIG. 6. The data presented in Table 2 demonstrate that there is a
statistically significant difference between the median values of the partition coefficients
of albumin in serum from patients with early (stage 1) breast cancer and those corresponding
to albumin in serum from patients with benign breast tumor. Furthermore, while the
separation between the two groups is not perfect, as in any diagnostics technology,
it nevertheless clearly provides means to clinically distinguish between the two phenotypes
with appropriate specificity and sensitivity levels selected for the desired clinical
application. Additionally, the data in Table 2 also indicate also that confounding
personal history is not related to the value of the partition coefficient. Finally,
statistical techniques such as Receive-Operating Characteristics analysis could be
performed (FIG. 7) and used to determine proper cut-off values of the K statistic.
As an example, selecting K = 1.39 provides for 100% sensitivity with 90% specificity
for the test.
Table 2. Partition coefficients for albumin in various serum samples from patients
with diagnostic status as indicated in the aqueous two-phase system PEG-1000-Ficoll-70-NaCl-potassium
citrate buffer, pH 6.8.
Patients with early stage breast cancer |
Patients with negative breast tumor biopsy |
Age |
Pathology |
Personal history |
K-value |
Error |
Age |
Personal history |
K-value |
Error |
34 |
G3; T1N0M0 |
chronic gastritis |
1.25 |
0.063 |
19 |
no |
1.62 |
0.033 |
55 |
G2; T1N0M0 |
chronic cystitis |
1.16 |
0.077 |
55 |
hypertension |
1.41 |
0.069 |
63 |
G3; T1N0M0 |
No |
1.24 |
0.053 |
28 |
No |
1.70 |
0.085 |
63 |
G1; T1N0M0 |
ischemia; hypertension |
1.26 |
0.054 |
59 |
ischemia; atherosclerosis |
1.89 |
0.052 |
67 |
G3; T1N0M0 |
Ischemia |
1.12 |
0.051 |
49 |
Hypertension |
1.51 |
0.039 |
46 |
G2; T1N0M0 |
No |
1.25 |
0.065 |
21 |
No |
1.30 |
0.084 |
83 |
G2; T1N0M0 |
ischemia; atherosclerosis |
1.21 |
0.057 |
38 |
chronic bronchitis |
1.47 |
0.088 |
38 |
G3; T1N0M0 |
No |
1.16 |
0.023 |
55 |
Hypertension |
1.88 |
0.042 |
84 |
G2; T1N0M0 |
ischemia; hypertension |
1.23 |
0.028 |
30 |
no |
1.54 |
0.029 |
56 |
G2; T1N0M0 |
cholelithiasis |
1.22 |
0.070 |
73 |
ischemia; atherosclerosis |
1.49 |
0.018 |
69 |
G2; T1N0M0 |
ischemia; atherosclerosis |
1.38 |
0.047 |
31 |
No |
1.80 |
0.089 |
65 |
G1; T1N0M0 |
Ischemia |
1.19 |
0.087 |
27 |
No |
12.0 |
0.11 |
66 |
G2; T1N0M0 |
Hypertension |
1.14 |
0.050 |
48 |
bronchitis; chronic |
1.54 |
0.042 |
|
|
|
|
|
|
gastritis |
|
|
56 |
G3; T1N0M0 |
Hypertension |
1.34 |
0.029 |
47 |
No |
1.23 |
0.046 |
60 |
G3; T1N0M0 |
ischemia; hypertension |
1.1 |
0.41 |
41 |
No |
1.52 |
0.029 |
|
|
|
|
|
49 |
No |
1.40 |
0.075 |
|
|
|
|
|
44 |
No |
2.1 |
0.12 |
|
|
|
|
|
32 |
No |
1.55 |
0.078 |
|
|
|
|
|
58 |
No |
1.9 |
0.16 |
|
|
|
|
|
39 |
No |
1.94 |
0.096 |
EXAMPLE 3
[0118] In this example it was demonstrated that the partition coefficients of albumin in
human serum from different patients with early (stage I) breast cancer are significantly
different from the partition coefficients of albumin in human serum from different
patients with benign breast tumor and patients with various malignant or benign tumors
of different tissue origins, thus demonstrating potential tissue-selectivity of a
test using the present invention.
[0119] Human serum samples corresponding to malignant and benign or healthy clinical phenotypes
were purchased from SeraCare Life Sciences. The diagnostic status of each sample was
provided by SeraCare Life Sciences. Samples aliquots were obtained frozen and stored
at - 80° C, and thawed and diluted 3-fold with water before introducing to the aqueous
two phase systems.
[0120] Aqueous two-phase system was prepared with PEG-1000 (polyethylene glycol with molecular
weight of about 1,000), Ficoll-70 (molecular weight of about 70,000), and potassium
citrate buffer, pH 6.8. The system in each tube was prepared by mixing the appropriate
amounts of stock polymer and buffer solutions dispensed by liquid handling workstation
Hamilton ML-4000 into a microtube of a total volume of 1.2 mL up to a total volume
of a mixture of 420 microliters. A varied amount (20, 30, 40, 50, 60, and 70 microliters)
of each serum sample and the corresponding amount (60, 50, 40, 30, 20, and 10 microliters)
of water were added to a system. The ratio between the volumes of the two phases of
each system of a final volume of 500 microliters was as 1:1. The system was vigorously
shaken and centrifuged for 60 min at 3500 g in a refrigerated centrifuge with a microplate
rotor with the temperature maintained at 23 °C to speed the phase settling. Microtubes
were taken out of the centrifuge, and aliquots of 40 microliters from the top and
the bottom phases were withdrawn in duplicates and each diluted and mixed with appropriate
reagents as indicated below and used for further analysis performed.
[0121] Immunoassay analysis was conducted with aliquots (each of 40 microliters volume)
from the top and the bottom phases were diluted 10-fold with 0.9 wt. % NaCl solution
in water. The diluted aliquots were mixed by shaking and 200 microliters from each
diluted aliquot was transferred to cups of clinical chemistry analyzer (Roche-Hitachi,
model 902). The turbidity-based microalbumin immunoassay was performed according to
the manufacturer's (Roche) protocol, and the albumin concentration in each aliquot
was recorded. The measured albumin concentration values in diluted aliquots from the
top phases were plotted as a function of the albumin concentration values of the similarly
diluted aliquots from the bottom phases. The partition coefficient for albumin was
determined as a slope of the linear curve.
[0122] The partition coefficients for albumin are presented in Table 3 (below). The means
for each group of samples corresponding to the same disease phenotype are plotted
in FIG. 8. The data demonstrate that it was possible to develop an appropriate chemical
composition of aqueous partitioning system that could differentiate between breast
cancer samples, and those corresponding to either benign or normal samples, or other
benign or cancer samples of different types of cancers. Furthermore, this particular
chemical composition was designed to validate a negative (benign) diagnosis made,
e.g., by mammography. Such a differential diagnosis with patients presenting suspicious
imaging results are often sent to invasive biopsies and/or other expensive procedures.
Other chemical compositions of aqueous partitioning systems could be developed for,
e.g., denote only cancerous condition of the breast with other cancers and benign
conditions of the breast resulting in significantly different K values.
Table 3. Partition coefficients for albumin in various serum samples from patients
with diagnostic status as indicated in the aqueous two-phase system PEG-1000-Ficoll-70-potassium
citrate buffer, pH 6.8 (BC-breast cancer; BB - breast benign.
Age |
Pathology |
K-value |
Error +/- |
Age |
Pathology |
K-value |
Error +/- |
80 |
BC; T1N0M0 |
3.07 |
0.04 |
68 |
BB |
3.31 |
0.071 |
45 |
BC, Stage 1 |
3.4 |
0.24 |
48 |
BB |
4.8 |
0.27 |
68 |
BC, Stage 1 |
3.09 |
0.06 |
42 |
BB |
5.1 |
0.13 |
42 |
BC, Stage 1 |
3.63 |
0.053 |
40 |
BB |
4.0 |
0.2 |
78 |
BC, Stage 1 |
3.13 |
0.046 |
26 |
BB |
4.5 |
0.12 |
42 |
BC, Stage 1 |
2.8 |
0.16 |
28 |
BB |
4.08 |
0.075 |
91 |
BC, Stage 1 |
3.36 |
0.044 |
35 |
BB |
3.98 |
0.021 |
n/a |
BC, Stage 1 |
2.6 |
0.12 |
41 |
BB |
4.6 |
0.3 |
n/a |
BC, Stage 1 |
2.92 |
0.034 |
31 |
BB |
4.0 |
0.11 |
n/a |
BC, Stage 1 |
2.99 |
0.034 |
44 |
BB |
4.26 |
0.021 |
n/a |
BC, Stage 1 |
3.37 |
0.071 |
56 |
BB |
4.1 |
0.05 |
n/a |
BC, Stage 1 |
2.98 |
0.05 |
41 |
BB |
3.24 |
0.075 |
n/a |
BC, Stage 1 |
3.18 |
0.046 |
n/a |
Healthy |
4.15 |
0.037 |
34 |
BC; G3; T1N0M0 |
3.4 |
0.15 |
n/a |
Healthy |
4.07 |
0.074 |
55 |
BC; G2; T1N0M0 |
3.62 |
0.051 |
n/a |
Healthy |
4.07 |
0.064 |
63 |
BC; G3; T1N0M0 |
3.0 |
0.13 |
67 |
Healthy |
4.07 |
0.095 |
63 |
BC; G1; T1N0M0 |
3.1 |
0.04 |
52 |
Healthy |
4.6 |
0.17 |
67 |
BC; G3; T1N0M0 |
2.79 |
0.038 |
19 |
BB |
4.5 |
0.2 |
46 |
BC; G2; T1N0M0 |
2.59 |
0.075 |
55 |
BB |
3.9 |
0.44 |
83 |
BC; G1; T1N0M0 |
2.75 |
0.077 |
28 |
BB |
4.4 |
0.25 |
73 |
BC; G3; T1N0M0 |
3.6 |
0.14 |
59 |
BB |
4.6 |
0.2 |
38 |
BC; G3; T1N0M0 |
3.09 |
0.032 |
49 |
BB |
5.1 |
0.21 |
84 |
BC; G2; T1N0M0 |
3.51 |
0.098 |
21 |
BB |
3.23 |
0.084 |
56 |
BC; G2; T1N0M0 |
3.4 |
0.14 |
38 |
BB |
2.9 |
0.14 |
69 |
BC; G2; T1N0M0 |
3.33 |
0.088 |
55 |
BB |
5.9 |
0.12 |
65 |
BC; G1; T1N0M0 |
3.5 |
0.15 |
30 |
BB |
3.9 |
0.14 |
66 |
BC; G2; T1N0M0 |
3.40 |
0.082 |
73 |
BB |
3.9 |
0.12 |
60 |
BC; G3; T1N0M0 |
3.10 |
0.071 |
31 |
BB |
4.1 |
0.23 |
56 |
BC; G3; T1N0M0 |
3.7 |
0.15 |
27 |
BB |
3.9 |
0.12 |
55 |
BC; G3; T1N0M0 |
3.4 |
0.17 |
48 |
BB |
4.4 |
0.18 |
55 |
BC; G2; T1N0M0 |
2.7 |
0.11 |
47 |
BB |
2.42 |
0.042 |
64 |
BC; G2; T1N0M0 |
2.48 |
0.025 |
41 |
BB |
3.6 |
0.11 |
60 |
BC; G2; T1N0M0 |
2.63 |
0.072 |
49 |
BB |
2.56 |
0.072 |
47 |
BC; G2; T1N0M0 |
2.99 |
0.085 |
44 |
BB |
2.54 |
0.054 |
48 |
BC; G2; T1N0M0 |
3.08 |
0.063 |
32 |
BB |
5.7 |
0.23 |
46 |
BC; G2; T1N0M0 |
3.1 |
0.2 |
50 |
BB |
4.34 |
0.092 |
75 |
BC; G2; T1N0M0 |
3.63 |
0.066 |
62 |
BB |
3.72 |
0.061 |
|
|
|
|
48 |
BB |
2.6 |
0.048 |
62 |
Colon Cancer |
4.2 |
0.25 |
50 |
BB |
3.70 |
0.098 |
71 |
Colon Cancer |
4.2 |
0.14 |
65 |
BB |
3.6 |
0.11 |
78 |
Colon Cancer |
4.4 |
0.2 |
58 |
BB |
4.5 |
0.11 |
69 |
Colon Cancer |
4 |
0.16 |
39 |
BB |
4.5 |
0.15 |
73 |
Colon Cancer |
4.1 |
0.22 |
|
|
|
|
85 |
Colon Cancer |
4.2 |
0.69 |
42 |
Lung Cancer |
4.5 |
0.18 |
|
|
|
|
57 |
Lung Cancer |
4.3 |
0.27 |
68 |
Colon Benign |
4.3 |
0.55 |
49 |
Lung Cancer |
4.3 |
0.29 |
71 |
Colon Benign |
6.0 |
0.61 |
64 |
Lung Cancer |
4.4 |
0.8 |
75 |
Colon Benign |
4.3 |
0.3 |
68 |
Lung Cancer |
4.1 |
0.15 |
69 |
Colon Benign |
4.8 |
0.23 |
|
|
|
|
74 |
Colon Benign |
4.8 |
0.1 |
72 |
Pancreatic cancer |
4.0 |
0.14 |
78 |
Colon Benign |
4.1 |
0.23 |
74 |
Pancreatic cancer |
4.4 |
0.14 |
|
|
|
|
69 |
Pancreatic cancer |
4.0 |
0.2 |
72 |
Prostate cancer |
4.13 |
0.075 |
81 |
Pancreatic |
3.97 |
0.094 |
|
|
|
|
|
cancer |
|
|
69 |
Prostate cancer |
4.9 |
0.17 |
|
|
|
|
81 |
Prostate cancer |
4.7 |
0.47 |
|
|
|
|
88 |
Prostate cancer |
4.32 |
0.085 |
|
|
|
|
74 |
Prostate cancer |
4.14 |
0.035 |
|
|
|
|
68 |
Prostate cancer |
4.4 |
0.38 |
|
|
|
|
71 |
Prostate cancer |
4.8 |
0.3 |
|
|
|
|
87 |
Prostate cancer |
5.2 |
0.33 |
|
|
|
|
|
|
|
|
|
|
|
|
69 |
Prostate Benign |
5.9 |
0.45 |
|
|
|
|
EXAMPLE 4
[0123] In this example it is demonstrated that the present invention could also be used
to differentiate physiological conditions other than cancer. In this example it is
shown that the partition coefficients of albumin in human serum from different patients
with nonalcoholic fatty liver disease (NAFLD) are significantly different from the
partition coefficients of albumin in human serum from healthy donors.
[0124] Human serum samples were purchased from SeraCare Life Sciences and PromedDx, Inc.
Samples were obtained frozen and were aliquoted and stored at -80 °C. Diagnostic status
of patients was provided by both vendors. Samples aliquots were thawed, brought to
the room temperature, and diluted 3-fold with water before introducing to aqueous
two phase systems.
[0125] The aqueous two-phase system prepared with PEG-1000 (polyethylene glycol with molecular
weight of about 1,000), Ficoll-70 (molecular weight of about 70,000), NaCl and potassium
citrate buffer, pH 6.8. Each system was prepared by mixing the appropriate amounts
of stock polymer and buffer solutions dispensed by liquid handling workstation Hamilton
ML-4000 into a microtube of a total volume of 1.2 mL up to a total volume of a mixture
of 440 microliters. A varied amount (10, 25, and 40 microliters) of each serum sample
and the corresponding amount (30, 15, and 0 microliters) of water were added to a
system. The ratio between the volumes of the two phases of each system of a final
volume of 480 microliters was as 1:1. The system was vigorously shaken and centrifuged
for 60 min at 3500 g in a refrigerated centrifuge with a microplate rotor with the
temperature maintained at 23 °C to speed the phase settling. Microtubes were taken
out of the centrifuge, and aliquots of 40 microliters from the top and the bottom
phases were withdrawn in duplicates and each diluted and mixed with appropriate reagents
as indicated below and used for further analysis performed as described below.
[0126] The other aqueous two-phase system contained 19.2 wt. % PEG-1000 (polyethylene glycol
with molecular weight of about 1,000), 5.8 wt. % Ficoll-70 (molecular weight of about
70,000), 0.10 M Na
2SO
4. and 18.2 wt. % potassium citrate buffer, pH 6.8. Each system was prepared by mixing
the appropriate amounts of stock polymer and buffer solutions dispensed by liquid
handling workstation Hamilton ML-4000 into a microtube of a total volume of 1.2 mL
up to a total volume of a mixture of 440 microliters. A varied amount (10, 25, and
40 microliters) of each serum sample and the corresponding amount (30, 15, and 0 microliters)
of water were added to a system. The ratio between the volumes of the two phases of
each system of a final volume of 480 microliters was as 1:1. The system was vigorously
shaken and centrifuged for 60 min at 3500 g in a refrigerated centrifuge with a microplate
rotor with the temperature maintained at 23 °C to speed the phase settling. Microtubes
were taken out of the centrifuge, and aliquots of 40 microliters from the top and
the bottom phases were withdrawn in duplicates and each diluted and mixed with appropriate
reagents as indicated below and used for further analysis performed as described below.
[0127] For immunoassay analysis aliquots (each of 40 microliters volume) from the top and
the bottom phases were diluted 10-fold with 0.9 wt. % NaCl solution in water. The
diluted aliquots were mixed by shaking and 200 microliters from each diluted aliquot
was transferred to cups of clinical chemistry analyzer (Roche-Hitachi, model 902).
The turbidity-based microalbumin immunoassay was performed according to the manufacturer
(Roche) protocol, and the albumin concentration in each aliquot was registered. The
measured albumin concentration values in diluted aliquots from the top phases were
plotted as a function of the albumin concentration values of the similarly diluted
aliquots from the bottom phases. The partition coefficient for albumin was determined
as a slope of the linear curve representing the slope.
[0128] The partition coefficients for albumin in examined serum samples from patients with
diagnostic status indicated are presented in Table 4. The data presented in Table
4 demonstrate that there is a significant difference between partition coefficients
of albumin in serum from patients with NAFLD and partition coefficients of albumin
in serum from healthy donors.
Table 4. Partition coefficients for albumin in serum samples from patients with diagnostic
status indicated in aqueous two-phase system prepared with PEG-1000, Ficoll-70, potassium
citrate buffer, pH 6.8 with different salts additives.
Salt additive |
Partition coefficient K for albumin in serum from patients with |
|
Healthy donors |
NAFLD |
Na2SO4 |
5.7 |
0.46 |
3.5 |
0.36 |
|
5.5 |
0.32 |
3.6 |
0.52 |
|
|
|
3.2 |
0.23 |
|
|
|
|
|
M NaCl |
1.5 |
0.21 |
0.96 |
0.089 |
|
1.4 |
0.32 |
0.93 |
0.060 |
|
|
|
0.81 |
0.012 |
[0129] Unless otherwise defined, all technical and/or scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to which
the invention pertains. Terms such as "biomolecule," "ligand," "cancer," breast cancer,"
"two-phase partitioning system," "ELISA," and other terms may have their normal meanings
in the art, unless otherwise stated.
[0130] As used herein the term "about" refers to +/- 10 %.
[0131] The term "partitioning system" or "biphasic system" or alike refer to a liquid system,
commonly with predominantly aqueous base and with a combination of soluble polymers
of various molecular weights such as poly-ethylene-glycol, dextran, ficoll, etc.,
and other additives, including salts, surfactants, etc. A partitioning system may
be prepared to physically result in phase separation, meaning that at least two distinct
phases appear with markedly different solvent properties, such as relative hydrophobicity,
ionic composition, etc.
[0132] As used herein, the singular form "a", "an" and "the" include plural references unless
the context clearly dictates otherwise. For example, the term "a compound" or "at
least one compound" may include a plurality of compounds, including mixtures thereof.
[0133] Throughout this application, various embodiments of this invention may be presented
in a range format. It should be understood that the description in range format is
merely for convenience and brevity and should not be construed as an inflexible limitation
on the scope of the invention. Accordingly, the description of a range should be considered
to have specifically disclosed all the possible subranges as well as individual numerical
values within that range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed subranges such as from 1 to 3,
from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual
numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless
of the breadth of the range.
[0134] Whenever a numerical range is indicated herein, it is meant to include any cited
numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges
between" a first indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are used herein interchangeably
and are meant to include the first and second indicated numbers and all the fractional
and integral numerals there between.
[0135] All technical terms may have their normal meaning as applied to the art unless otherwise
specified.
[0136] While several embodiments of the present invention have been described and illustrated
herein, those of ordinary skill in the art will readily envision a variety of other
means and/or structures for performing the functions and/or obtaining the results
and/or one or more of the advantages described herein, and each of such variations
and/or modifications is deemed to be within the scope of the present invention. More
generally, those skilled in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be exemplary and that
the actual parameters, dimensions, materials, and/or configurations will depend upon
the specific application or applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to ascertain using
no more than routine experimentation, many equivalents to the specific embodiments
of the invention described herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within the scope of the
appended claims and equivalents thereto, the invention may be practiced otherwise
than as specifically described and claimed. The present invention is directed to each
individual feature, system, article, material, kit, and/or method described herein.
In addition, any combination of two or more such features, systems, articles, materials,
kits, and/or methods, if such features, systems, articles, materials, kits, and/or
methods are not mutually inconsistent, is included within the scope of the present
invention.
[0137] All definitions, as defined and used herein, should be understood to control over
dictionary definitions, definitions in documents incorporated by reference, and/or
ordinary meanings of the defined terms.
[0138] The indefinite articles "a" and "an," as used herein in the specification and in
the claims, unless clearly indicated to the contrary, should be understood to mean
"at least one."
[0139] The phrase "and/or," as used herein in the specification and in the claims, should
be understood to mean "either or both" of the elements so conjoined, i.e., elements
that are conjunctively present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the same fashion, i.e.,
"one or more" of the elements so conjoined. Other elements may optionally be present
other than the elements specifically identified by the "and/or" clause, whether related
or unrelated to those elements specifically identified. Thus, as a non-limiting example,
a reference to "A and/or B", when used in conjunction with open-ended language such
as "comprising" can refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally including elements other
than A); in yet another embodiment, to both A and B (optionally including other elements);
etc.
[0140] As used herein in the specification and in the claims, "or" should be understood
to have the same meaning as "and/or" as defined above. For example, when separating
items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a number or list of
elements, and, optionally, additional unlisted items. Only terms clearly indicated
to the contrary, such as "only one of' or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element of a number or
list of elements. In general, the term "or" as used herein shall only be interpreted
as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one
of." "Consisting essentially of," when used in the claims, shall have its ordinary
meaning as used in the field of patent law.
[0141] As used herein in the specification and in the claims, the phrase "at least one,"
in reference to a list of one or more elements, should be understood to mean at least
one element selected from any one or more of the elements in the list of elements,
but not necessarily including at least one of each and every element specifically
listed within the list of elements and not excluding any combinations of elements
in the list of elements. This definition also allows that elements may optionally
be present other than the elements specifically identified within the list of elements
to which the phrase "at least one" refers, whether related or unrelated to those elements
specifically identified. Thus, as a non-limiting example, "at least one of A and B"
(or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or
B") can refer, in one embodiment, to at least one, optionally including more than
one, A, with no B present (and optionally including elements other than B); in another
embodiment, to at least one, optionally including more than one, B, with no A present
(and optionally including elements other than A); in yet another embodiment, to at
least one, optionally including more than one, A, and at least one, optionally including
more than one, B (and optionally including other elements); etc.
[0142] When the word "about" is used herein in reference to a number, it should be understood
that still another embodiment of the invention includes that number not modified by
the presence of the word "about."
[0143] It should also be understood that, unless clearly indicated to the contrary, in any
methods claimed herein that include more than one step or act, the order of the steps
or acts of the method is not necessarily limited to the order in which the steps or
acts of the method are recited.
[0144] In the claims, as well as in the specification above, all transitional phrases such
as "comprising," "including," "carrying," "having," "containing," "involving," "holding,"
"composed of," and the like are to be understood to be open-ended, i.e., to mean including
but not limited to. Only the transitional phrases "consisting of' and "consisting
essentially of' shall be closed or semi-closed transitional phrases, respectively,
as set forth in the United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
[0145] The current disclosure contains,
inter alia, the following items:
- 1. A method for diagnosing breast cancer comprised of the following:
collecting blood from a patient;
separating serum or plasma from said blood;
partitioning said serum or plasma in at least one aqueous two-phase partitioning system;
assaying aqueous phases of said at least one two phase partitioning system for human
serum albumin (HSA) using at least one assay specific for said albumin;
calculating partition coefficient K of albumin for each aqueous phase of said two
phase partitioning system; and,
determining presence, lack of, or risk level of breast cancer in said patient by comparing
numerical values of calculated partition coefficients with reference values previously
determined for HSA in serum or plasma taken from individuals with and without breast
cancer.
- 2. The method according to item 1, wherein said assaying is performed with an HSA-specific
immuno-based assay.
- 3. The method according to any one of items 1 or 2, wherein said assaying is performed
with an assay that is specific for a biomolecule that is associated with HSA.
- 4. The method according to any one of items 1-3, wherein said two-phase partitioning
system is adapted to differentially partition albumin when breast cancer is present
or absent.
- 5. The method according to any one of items 1-4, wherein said method is applied in
conjunction with another breast cancer test.
- 6. The method according to any one of items 1-5, wherein said method is applied as
a part of a mathematical or statistical algorithm, in conjunction with information
obtained from another breast cancer test.
- 7. The method according to any one of items 1-6, wherein said individuals without
breast cancer include individuals with benign tumors.
- 8. The method according to any one of items 1-7, wherein said diagnosing is used to
screen, diagnose, classify according to phenotype/genotype, aid in therapeutic course
of action, monitor progression, or detect recurrence of breast cancer.
- 9. The method according to any one of items 1-8, wherein said numerical value of the
partition coefficient is used to select a therapeutic drug.
- 10. The method according to any one of items 1-9, wherein said partitioning comprises
vortexing and centrifugation of said two-phase partitioning system with human serum
albumin.
- 11. The method according to any one of items 1-10, further including the step of removing
at least one biomolecule from said human serum albumin.
- 12. The method according to item 11, wherein said at least one biomolecule is characterized
for use as a biomarker for breast cancer presence or risk.
- 13. The method according to any one of items 1-12, wherein said reference values are
determined from blood samples taken from individuals with and free of breast cancer.
- 14. A device for the detection of breast cancer, including:
a unit for collecting blood from a patient and separating serum or plasma from said
blood;
at least one aqueous two-phase partitioning system in fluid communication with the
unit for collecting blood;
a unit for partitioning a portion of said serum or plasma in said two phase partitioning
system in fluid communication with the partitioning system;
an assay for determining the presence of human serum albumin in aqueous phases of
said two phase partitioning system;
a computing element adapted to determine a coefficient K, wherein K represents the
distribution of human serum albumin in the aqueous portions of said two-phase partitioning
system; and,
a determination element adapted to compare said coefficient K with known values of
K for blood samples of healthy individuals and individuals with breast cancer.
- 15. The device according to item 14, wherein said assay is an HSA-specific immuno-based
assay.
- 16. The device according to any one of items 14 or 15, wherein the device comprises
a microfluidic channel in fluidic communication with the partitioning system.
- 17. The device according to any one of items 14-16, wherein said two-phase partitioning
system is adapted to differentially partition albumin when breast cancer is present
or absent in said patient.
- 18. The device according to any one of items 14-17, wherein said computing element
and said determination element are a single element.
- 19. The device according to item 18, wherein said computing device is one of the following:
mainframe computer, laptop computer, tablet computer, mobile computing device, and
tabletop computer.
- 20. The device according to any one of items 14-19, wherein said unit for partitioning
comprises a centrifuge component.
- 21. The device according to any one of items 14-20, wherein the patient is human.
- 22. A method for diagnosing a disease in a patient comprised of the following:
collecting blood from said patient;
separating serum or plasma from said blood;
partitioning said serum or plasma in at least one aqueous two-phase partitioning system,
wherein said two-phase partitioning system is adapted to differentially partition
albumin when said disease is present or absent in said patient;
assaying aqueous phases of said at least one two phase partitioning system for human
serum albumin (HSA) using specific assay for said albumin;
calculating partition coefficient K of albumin for each aqueous phases; and,
determining presence of said disease in said patient by comparing numerical values
of calculated partition coefficients with reference values previously determined for
albumin in serum or plasma taken from individuals with and without said disease.
- 23. The method according to item 22, wherein said disease is cancer.
- 24. The method according to item 23, wherein said cancer is selected from the following:
lymphomas, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
synovioma, mesothelioma, lymphangioendotheliosarcoma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat
gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, testicular tumor, lung carcinoma, non-small cell lung carcinoma,
small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias,
acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic leukemia, promyelocytic
leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia,
chronic myelocytic leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma,
Hodgkin's disease, multiple myeloma, or Waldenstrom's macroglobulinemia.
- 25. The method according to any one of items 22-24, wherein said disease is a plurality
of diseases.
- 26. The method according to any one of items 22-25, wherein said disease is hereditary.
- 27. The method according to any one of items 22-26, wherein the partition coefficients
obtained from a plurality of aqueous two-phase systems are combined using mathematical
techniques into a numerical signature.
- 28. The method according to item 27, wherein said numerical signature is compared
with numerical signatures obtained from reference values and is used for diagnosis.
- 29. The method according to any one of items 22-28, wherein said diagnosis is performed
by comparing the value of the partition coefficient, K, to its prior value or values
at prior time or times of the same individual.
- 30. A method, comprising:
partitioning a sample arising from a subject in an aqueous multi-phase partitioning
system; and
determining the distribution of human serum albumin within the phases of the partitioning
system.
- 31. The method of item 30, comprising determining human serum albumin within only
one phase of the partitioning system.
- 32. The method of any one of items 30 or 31, comprising determining human serum albumin
within each phase of the partitioning system.
- 33. The method of any one of items 30-32, further comprising comparing the distribution
of human serum albumin to a reference distribution.
- 34. The method of item 33, wherein the reference distribution is an average distribution
of a population of normal humans.
- 35. The method of any one of items 33 or 34, wherein the reference distribution is
the distribution of human serum albumin of the subject at a different point in time.
- 36. The method of any one of items 30-35, further comprising determining a disease
state of the subject based on the distribution of human serum albumin.
- 37. The method of item 36, wherein the disease state is cancer.
- 38. The method of item 37, wherein the disease state is breast cancer.
- 39. The method of any one of items 30-38, comprising determining a partition coefficient
for human serum albumin in the partitioning system.
- 40. The method of any one of items 30-39, wherein the sample is a blood sample.
- 41. The method of item 40, further comprising producing serum from the blood sample.
- 42. The method of item 40, further comprising producing plasma from the blood sample.
- 43. The method of any one of items 30-39, wherein the sample is a serum sample.
- 44. The method of any one of items 30-39, wherein the sample is a plasma sample.
- 45. The method of any one of items 30-44, wherein determining the distribution of
human serum albumin comprises assaying at least one phase of the partitioning system
using an immunoassay.
- 46. The method of any one of items 30-45, further comprising selecting a drug for
administration to the subject based on the distribution of human serum albumin.
- 47. The method of any one of items 30-46, wherein the aqueous multi-phase partitioning
system is an aqueous two-phase partitioning system.